Hematopoietic stem cells (HSC) are defined as clonogenic cells that can both self-renew and give rise to all blood cell lineages. In the past grant period we demonstrated a skewing in blood cell lineage representation as mice and humans age, from a balance between lymphopoiesis and myelomonocytopoiesis in young adult life, to a predominance of myelomonocytopoiesis in the elderly. This aging-related skewing of hematopoietic differentiation outcome potentially explains the increased susceptibility late in life to myeloid malignancies and anemia, as well as diminished adaptive immunity to infectious disease. In this proposal, we consider two possible mechanisms for this aging-related phenomenon (1) that all HSC clonotypes in young adult life produce a balanced repertoire of blood cell lineages, but acquire a series of epigenetic changes over time which silence some genes and induce others, resulting in elderly HSC which are biased to myelomonocytopoiesis; or (2) that balanced and myeloid-biased HSC clonotypes both exist in young adults, with myeloid-biased HSC clonotypes being preferentially clonally selected over time by environmental, feed- back, and feed-forward regulatory external stimuli. While our preliminary data favor the latter explanation, the current proposal will definitively evaluate these alternatives by undertaking a comprehensive analysis of the diversity and maturation of different HSC clonotypes. We will initially determine the number of different kinds of HSC clones, using the single cell Biomark assay for diverse gene expression patterns, and evaluating HSCs obtained from embryonic, fetal, young adult, and aged mice. We will label individual HSCs of different types with sensitive clone marking techniques, then study their origin, maturation, migration, and regulation in mice. We will start in adults, tracing them back to the fetal or late embryonic stages of development. We will trace them forward to aging and to leukemogenesis. We will test whether the end blood cell types [RBC, platelets, granulocytes, monocytes and macrophages, the various lymphocytes] when lacking or transfused in overabundance bring endogenous HSC clones into or out of cell cycle to expand or diminish the number of certain HSC clonotypes. We will evaluate whether the same aging-related skewing of HSC differentiation outcomes occurs in humans. This examination of whether HSC clonal diversity is pre-determined by birth, with hematopoietic balance throughout life being shaped by clonal selection in response to diverse physiological events, is the theme that will drive the basic research of this grant.